Protein-protein interactions (PPI), which are the ultimate determinants of the function of all proteins, can rarely be modulated by small molecules because the corresponding protein interfaces do not have adequate binding pockets. Recently, an intriguing alternative has been recognized for constitutively multimeric proteins such as those in the important TNF superfamily (TNFSF) via an allosteric mechanism that distorts one of the binding partners. Since we have recently identified the first small-molecule inhibitors of the CD40CD154 costimulatory interaction, which is a member of this family, we propose to exploit the possible advantages that can be derived from such a mechanism and (1) confirm the feasibility of an allosteric, trimer-distorting mechanism in interfering with the CD40CD154 costimulatory proteinprotein interaction, a member of the TNFSF that plays an important role in the activation of immune responses, (2) use this information and design improved small-molecule inhibitors suitable for therapeutic applications and to confirm the immunosuppressive activity of the most promising inhibitors, and (3) investigate whether similar mechanism can be found for other members of the TNF superfamily (in particular, OX40OX40L, BAFF-RBAFF, RANKRANKL, 4-1BB4- 1BBL) and whether it can be used to identify specific inhibitors. TNFSF costimulatory interactions play key roles in the development of effective immune responses, but until relatively recently, they, just as most other PPIs, were considered undruggable. It has now been suggested that such inhibitions for CD40CD154 and other members of the TNFSF family might occur via a unique allosteric mechanism whereby the disruptor molecule intercalates not between the receptorligand interface, but between monomeric units of the trimeric ligand (or receptor). This can allow more efficient binding making these interactions particularly targetable by small molecule. Whereas drug-like chemical libraries commonly used for high-throughput screening are now recognized to not be well-suited for PPI inhibition, the chemical space of our inhibitors can allow quick exploration of this novel mechanism for constitutively homotrimeric cytokines, and can lead to novel pharmacological tools and new innovative drugs for the many disease areas where TNF superfamily interactions are involved. Development of a detailed mechanistic understanding of the small-molecule inhibition of CD40CD154 and possibly other receptor-ligand pairs of the TNF superfamily can lead to the development of novel, clinically feasible approaches addressing therapeutic needs arising from dysregulated functions of costimulatory molecules in T-, B-, and APC cells such as autoimmune diseases and transplant rejection. Accordingly, the proposed work should lead to the elucidation of an intriguing new mechanism that can make possible small-molecule costimulatory blockade as well as to novel pharmacological tools and new innovative drugs for a number of disease areas where TNF superfamily interactions are involved. Because of its unique combination of expertise in small molecule pharmacology, medicinal chemistry, molecular biology, cell transplant, and immunobiology, we believe that our team is very well positioned to achieve considerable progress along these lines.